Antibody Binding Capacity Research Articles

Abnormal platelet immunophenotypes and percentage of giant platelets in myelodysplastic syndrome: A pilot study.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic stem cell disorder with thrombocytopenia. Flow cytometric immunophenotyping of blood cells has been instrumental in diagnosis as co-criteria, but the data regarding platelets remains lacking. This study aims to determine if there is a difference in surface antigen levels on platelets by comparing surface antigen levels in MDS patients and healthy control subjects. Concurrently, as flow cytometric gating can reveal the diameter of cells, this study will investigate differences in giant platelet percentage by comparing these percentages in high- and low-risk MDS patients. Twenty newly diagnosed MDS patients were enrolled in this study. Platelet surface antigen levels were determined by measuring the binding capacity of antibodies with flow cytometry. Platelets of MDS patients were shown to have a lower level of CD61 and higher levels of CD31 and CD36 than healthy controls. Judged by forward scatter (FSC), MDS patients' platelets appeared to be larger than those of healthy control subjects, whereas the MFI adjusted by diameter (MFI/FSC ratio) of CD31, CD41a, CD42a, CD42b and CD61 on platelets were lower in MDS patients than in healthy control subjects. There was a significant quantity of giant platelets found in MDS patients, and the high-risk MDS patients tended to have a higher percentage of giant platelets than low-risk patients. Conclusions: All the results indicate that MDS patients exhibit a lower antigen presentation (MFI) adjusted by diameter on platelets than healthy controls and the giant platelets detected by flow cytometry might correlate with the condition of MDS.

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.

KLRG1 Depletion Is a Novel Therapeutic Strategy for Patients with Mature T and NK/T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory immune checkpoint receptor expressed on late differentiated CD8+ T effector memory (TEM) and TEMRA cells. Monotherapy with KLRG1 neutralizing antibodiesextended survival in solid tumor models. Here, we report the therapeutic potential of a novel and distinct strategy characterized by depletion of KLRG1+ malignant cells in hematologic malignancies and specifically in TCLs. We defined the molecular epidemiology of KLRG1 by determining tumor cell-specific RNA and protein expression on T cell subsets from 8 healthy donors, 26 TCL lines, 15 patient-derived xenografts (PDXs), and 165 tumor and blood specimens representing various subtypes of T and NK/TCLs. Microarray andRNA-seqdata demonstrated highertranscript levels of KLRG1 in patients with T-LGLL and PDXs of T-PLL and HSTCL relative to other subtypes, which were corroborated by protein expression. We quantified tumor cell-specific protein expression of KLRG1 by IHC by generating H scores (% of KLRG1+ tumor cells × measure of staining intensity) ranging from 0-300 across 141 primary TCL biopsies. PTCL-NOS and AITL had heterogeneous KLRG1 expression, whereas ALCL, CTCL, and EATL demonstrated none. The majority of patients with T-PLL (median 180), δ/γ TCLs (median 140), and NK/TCLs (median 100) exhibited higher H scores relative to other subtypes. Up to 99% of the CD3+/CD8+/CD57+ and up to 97% of the CD3-/CD56+/CD57+/CD16+/CD94+ leukemic cells in the blood of patients with T-LGLL (n=12) and CLPD-NK (n=12) respectively expressed KLRG1 protein (Fig.1). None of the TCL cell lines expressed surface KLRG1. Therefore, SMZ1 cells (TP63 rearranged PTCL-NOS line) were stably transduced to express KLRG1. We utilized two human KLRG1 binding antibodies, 13F12F2 (a commercial mIgG2a Ab) and mAb208 (a humanized afucosylated mIgG2a Ab) for in vitro and in vivo studies. Antibody binding capacity assays confirmed a higher number of KLRG1 receptors per cell on SMZ1 KLRG1+ (175,800), HSTCL (23,586), and CD8+ T cells (16,326) compared with B-cell lymphoma cells (242). Competition binding assays demonstrated that concentrations of 0.1 ug/ml of mAb208 saturated receptors on SMZ1 KLRG1+ cells. mAb208 induced a 2-5-fold dose-dependent increase in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), but not apoptosis of SMZ1 KLRG1+ cells relative to SMZ1 KLRG1- WT and isotype control (CTL). This was exhibited across a variety of human and murine effectors such as human PBMCs, human and murine macrophages, and human and murine complements. Reporter-based murine FcγRIV ADCC bioassay demonstrated a dose-dependent 2-6 fold induction of bioluminescence by mAb208 in SMZ1 KLRG1+ cells relative to WT (p=<0.0001). In 4/4 tested T-LGLL patient samples, mAb208 induced ADCC of CD8+/CD57+ LGLs with autologous PBMCs (p=0.03) relative to CTL. Finally, mAb208 induced selective ADCC of KLRG1+ CD8+ TEM and TEMRA cells while sparing KLRG1- naive and central memory T cells in 3 healthy donors (p=0.06). Duvelisib (Duv), a dual PI3K-δ/γ inhibitor (PI3Ki), has demonstrated a high ORR of 50% in phase II trials in TCLs and its ability to reprogram macrophages to an M1 phenotype in TCL PDXs. Thus, we hypothesized that the combination of PI3Ki with anti-KLRG1 mAbs would enhance the clearance of apoptotic and opsonized KLRG1+ TCL cells by proinflammatory macrophages in several PTCL subtypes. We tested this combination in vivo using SMZ1 KLRG1+ subcutaneous xenografts in NSG mice. After 17 days of treatment, combination (mAb208 + Duv)- treated mice had a marked reduction in their mean tumor volumes (159 mm3) relative to mAb208 (1003 mm3 ; p=0.03), Duv (465.6 mm3 ; p=0.03) and CTL (2104 mm3 ; p=0.03) mice resulting in prolonged survival (Fig. 2). Studies in PDX models of T-PLL and HSTCL with varying levels of KLRG1 expression (30% and 80% respectively) have been initiated and will be reported at the meeting. Selective depletion of tumor cells with an anti-KLRG1 depleting mAb in specific subtypes of TCLs, T-LGLL, and CLPD-NK is effective in vitro and in vivo, and its combination with Duv has marked activity in an aggressive PTCL-NOS xenograft model. A multicenter phase I/II trial of the KLRG1 depleting antibody ABC008 (Abcuro) in patients with R/R T-LGLL has been initiated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

The application of antibody-functionalized quantum dots (QDs) in different areas has been widely described in the literature. However, a standard routine method for obtaining information on the conjugation efficiency of QDs with antibodies in terms of the interaction of the functionalized QDs with a specific antigen is still lacking. Herein, surface plasmon resonance (SPR) spectroscopy is proposed for this purpose. Gold-coated SPR sensor disks were modified with a self-assembled monolayer of 11-mercaptoundecanoic acid, and carbodiimide cross-linker chemistry was used to covalently immobilize the CD44 biomarker on the premodified surface (Au/CD44). Meanwhile, QDs functionalized with amine-derivatized polyethylene glycol (PEG) (QDs-NH2) were chosen for conjugation with antibodies because of their low non-specific adsorption on the Au/CD44 surface. Prior to conjugation, the surface binding capacity (Bmax) and equilibrium dissociation constant (KD) of the specific antibodies against CD44 (anti-CD44) were found to be 263.32 ± 2.44 m° and 1.00 × 10−7 ± 2.29 × 10−9 M, respectively. QDs-NH2 and anti-CD44 were conjugated at their initial molar ratios of 1:3, 1:5, 1:10 and 1:12. SPR measurements showed that the conjugates (QDs-anti-CD44) prepared using 1:10 and 1:12 molar ratios interacted comparably with immobilized CD44 biomarkers. The equilibrium angles in the case of 10- and 12-fold concentrations of anti-CD44 were calculated to be 60.43 ± 4.51 and 61.36 ± 4.40 m°, respectively. This could be explained by the QDs-NH2 and anti-CD44 having a similar surface loading (about four molecules per QDs-NH2) and similar hydrodynamic diameters, which were 46.63 ± 3.86 and 42.42 ± 0.80 nm for the 1:10 and 1:12 ratios, respectively. An initial QDs-NH2: anti-CD44 molar ratio of 1:10 was chosen as being optimal. SPR spectroscopy proved to be the right choice for QDs-anti-CD44 conjugation optimization, and can be used for the evaluation of conjugation efficiency for other nanostructures with various bio-recognition molecules.

Abstract 1870: A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development

Abstract Receptor occupancy (RO) assays are used to quantify the binding of therapeutic antibodies to their targets on the cell surface. This method can be used to in early drug discovery to determine the antibody binding capacity (ABC) of drug candidates to cells in culture to hone the desired drug characteristics during candidate screening or lead optimization. This method can also be used in xenografts or patient tissues to create pharmacodynamic (PD) data that directs the efficacy hypothesis and biomarker selection for clinical trials. The current approaches use flow cytometry on fresh specimens, which introduces many technical and logistical challenges and does not retain spatial information about the tissue. Here we introduce a new, simple method which can be performed on Formalin Fixed, Paraffin Embedded (FFPE) cells or tissues. This approach eliminates the challenges of flow cytometry methods, provides the type of quantitative data needed, and maintains the tissue contexture to study the effects of immune-directing antibodies in patient tissue samples. We describe a method for developing a standard curve to determine antibody binding capacity (ABC) using our Quanticell ™ fluorescent nanoparticle technology. We demonstrate this in a typical bispecific antibody use case, where both the immune-directing (CD3) and targeting (Her2) arms of the bispecific are modulated to create the desired efficacy characteristics. We demonstrate the establishment of a standard calibration curve for Her2 (BT474/Her2+) and CD3 (Jurkat/CD3+) in cells with titrated amounts of anti-Her2 and anti-CD3 antibodies which target the extracellular domain necessary for these studies. We also show how the same Quanticell assay can be used in clinical tissue, to evaluate the localization of the drug targets and immune cells within the tissue context of human patient samples necessary to understand how pharmacodynamic properties of the drug can modulate immune contexture changes. The Quanticell™ method is a simple immunohistochemistry (IHC) based method which allows extremely sensitive detection of therapeutic antibodies bound on FFPE fixed cells or tissue, which thus does not require the use of flow cytometry in fresh tissues, allowing simple use from early discovery through clinical research studies. Importantly, the Quanticell™ method is capable of detecting a very large and linear dynamic range of analyte concentration to allow full quantification of ABC for RO studies. Critically, the Quanticell™ method retains the tissue context, allowing for simultaneous and spatial evaluation of multiple analytes such as the target of the antibody and the immune contexture. This method allows for a consistent method for RO studies that can be applied to early discovery, translational, and clinical research to optimize drug efficacy. Citation Format: Atsuro Tatsumi, Keisuke Morichika, Caroline Morel, Omid Ghasemi, Hiroyuki Yokota, Joseph S. Krueger. A simple and quantitative immunoassay for evaluation of receptor occupancy and antibody binding capacity in fixed cells or tissue samples for immune-directing antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1870.

Assessing the activity of antibodies conjugated to upconversion nanoparticles for immunolabeling

Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from individual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies.

Versatile and Easily Designable Polyester-Laser Toner Interfaces for Site-Oriented Adsorption of Antibodies.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0–100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

Actinidin-induced hydrolysis of milk proteins: Effect on antigenicity

Actinidin was used to hydrolyse proteins in whey protein isolate (WPI) and milk protein concentrate (MPC) to reduce immunoreactivity of β-lactoglobulin (β-LG) and αs1-casein (αs1-CN). Samples were incubated at an enzyme to substrate ratio of 1:100 (5.21 units of actinidin activity g−1 of protein) at 10 or 60 °C for up to 31 or 5 h, respectively. Protein hydrolysis was determined by the degree of hydrolysis and SDS-PAGE. Antigenicity of hydrolysates was determined by β-LG and αs1-CN antibody-binding capacity using enzyme-linked immunosorbent assay (ELISA) quantification kits. ELISA showed significant reduction of antigenicity of β-LG and αs1-CN with higher degree of hydrolysis (DH) by actinidin. At 60 °C, hydrolysis for 5 h resulted in antigenicity reduction of ∼43% for β-LG and ∼48% for αs1-CN in MPC and ∼54% for β-LG in WPI. Hydrolysis at 10 °C for 31 h also resulted in decrease in antigenicity in MPC for β-LG and αs1-CN by ∼39 and 42% respectively, but only 14% for β-LG in WPI. Treatment with actinidin can reduce the antigenicity by modification of protein conformation and cleavage and masking of epitopes of β-LG and αs1-CN.

Methods for Enhanced Fluorescence Detection of Proteins by using Entrapped Gold Nanoparticles in Membranes.

Measuring protein levels from biofluids can provide important insight into human health and disease during various physiological and pathological conditions. In many situations, sensitive methods are required for protein quantification because at the early stages of many diseases, proteins in biofluids are present at very low concentrations. Here, a new and simple method is presented in the form of Basic and Alternative Protocols for an immunoassay performed on a nitrocellulose membrane, followed by the addition of gold nanoparticles prior to measuring fluorescence with a microscope. The assay protocol was optimized to achieve 3D metal-enhanced fluorescence (MEF) with increased antibody-binding capacity and enhanced fluorescence signals, improving assay sensitivity. Using different concentrations of spiked fluorescently labeled IgGs in pooled normal human plasma, a lower detection limit of 29 ng/ml was achieved. This limit of detection was found to be a thousand-fold lower than the conventional 2D assay and one order of magnitude lower than when the assay was performed on a 3D membrane without MEF. This method provides an easy way to improve immunoassay sensitivity, and it can be simply transferred to other labs. It also can extend to fluorescence detection of other analytes beyond proteins. © 2022 Wiley Periodicals LLC. Basic Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using commercially available AuNPs Alternative Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using lab-made AuNPs.

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative.

BackgroundThe Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking.ObjectiveTo develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories.MethodsA high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation.ResultsUsing automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen.ConclusionWe optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

MRNA-1273 vaccine-induced antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization.

Entrapping gold nanoparticles in membranes for simple-to-use enhanced fluorescence detection of proteins

Quantifying proteins under different physiological and pathological conditions can give important insights into human health and disease, since proteins are the functional components of cells. In order to be able to use protein expression levels to diagnose diseases, sensitive protein quantification techniques are required, because some proteins can be present in low concentrations in biofluids at early stages of a disease. Here, a novel and simple-to-implement signal enhancement method for fluorescence-based protein detection is presented, in which an immunoassay was conducted on a nitrocellulose membrane, and a solution of gold nanoparticles was then pipetted onto the membrane before signal acquisition with a fluorescence microscope. The gold nanoparticles were entrapped and adsorbed into the 3D membrane matrix upon drying due to the hydrophobic interactions with the membrane. Through optimizing the concentration and size of the nanoparticles and comparison of different membranes, we were able to achieve metal enhanced fluorescence (MEF). This new gold nanoparticle-based MEF method, together with the 3D membrane platform that improves antibody and gold nanoparticle binding capacity, provided higher signal intensity and assay sensitivity for fluorescence-based protein quantification. Fluorescently-labeled IgG protein was spiked in human plasma with different concentrations, and the lower limit of detection was determined to be 29 ng/mL, three orders of magnitude lower than that of conventional 2D assays and one order of magnitude lower than that of 3D membrane assays without applying gold nanoparticles. This method offers a simple way of improving the sensitivity of fluorescence-based immunoassays that can be easily adopted in a biological lab with basic lab set up. Furthermore, it can be potentially extended to the fluorescence detection of other analytes beyond proteins.

Treatment of Patients with T Cells Expressing a Fully-Human Anti-BCMA CAR with a Heavy-Chain Antigen-Recognition Domain Caused High Rates of Sustained Complete Responses and Relatively Mild Toxicity

Multiple myeloma (MM) is a malignancy of plasma cells that is nearly always incurable. T cells expressing chimeric antigen receptors (CAR) that target B-cell maturation antigen (BCMA) can recognize and eliminate MM. The murine or other non-human sequences in the single-chain variable fragments (scFv) of many anti-BCMA CARs can elicit recipient immune responses against CAR T cells. We constructed a CAR incorporating an anti-BCMA fully-human heavy-chain variable domain designated FHVH33. FHVH33 lacks the light chain, the artificial linker sequence, and the 2 linker-associated junctions of a scFv, so FHVH33 is smaller than a scFv and is likely to be less immunogenic. The FHVH33-containing CAR utilized in this clinical trial also incorporated a CD8a hinge and transmembrane domain, a 4-1BB domain, and a CD3z domain. The CAR was designated FHVH33-CD8BBZ and was encoded by a gamma-retroviral vector. T cells expressing FHVH33-CD8BBZ were designated FHVH33-T. The FHVH33-T production process was initiated with unsorted peripheral blood mononuclear cells and took 7 days. The treatment protocol was 300 mg/m 2 of cyclophosphamide and 30 mg/m 2 of fludarabine on days -5 to -3 followed by infusion of FHVH33-T on day 0.Twenty-five patients received FHVH33-T infusions. Median age of the treated patients was 62 (range 39-73). Patients received a median of 6 prior lines of therapy (range 3-10). Five dose levels were assessed (Table). Dose level 4, 6x10 6 CAR + T cells/kg was identified as the maximum feasible dose after considering efficacy and manufacturing factors.Twenty-three of 25 patients (92%) obtained objective responses (OR) of partial response (PR) or better. Seventeen patients (68%) attained a best response of stringent complete response (sCR) or very good partial response (VGPR). Thirteen patients have ongoing responses. To date, the median duration of response is 50 weeks for the highest two dose levels. At present, the overall median progression free survival (PFS) is 78 weeks; as responses are ongoing in 13 patients (52%), PFS will likely improve. Nine of 25 patients had extramedullary plasmacytomas at baseline; patients with extramedullary plasmacytomas at baseline were less likely to achieve sCR (P=0.011).All 25 treated patients were evaluable for toxicity. Eighteen patients had grade 1 or 2 cytokine-release syndrome (CRS), and 6 patients had grade 3 CRS. One patient had no CRS. No patients had grade 4 CRS. Five patients received tocilizumab and 4 patients received corticosteroids for CRS. Two of twenty-five patients had grade 3 neurological toxicity possibly attributable to FHVH33-T. No patient had grade 4 neurologic toxicity attributable to CAR T cells. One patient died of influenza pneumonia.We assessed blood CAR+ cells by quantitative PCR. The median peak blood CAR+ cell level was 126.5 cells/µl (range 3-1071 cells/µl), and the median time post-infusion of peak blood CAR + cell levels was 10.5 days (range 7-14). Peak CAR T-cell level was not associated with obtaining a sCR. In contrast, blood CAR+ T cell levels at both 1 and 2 months after infusion were statistically higher for patients obtaining sCR. For the 1-month time-point, blood CAR+ cell levels in cells/mL were 20 for sCR patients and 4 for not sCR patients (P=0.04). Pretreatment serum BCMA was not statistically different when patients obtaining or not obtaining sCR were compared (median serum BCMA in pg/mL: sCR patients 86,243; not sCR patients 261,675, P=0.20). We assessed cell-surface BCMA expression level on MM cells by antibody binding capacity (ABC) flow cytometry. Cell-surface BCMA expression level was not statistically different in sCR versus not sCR patients (median ABC in sites/cell: sCR patients 844; not sCR patients 535, P=0.29). Patients with MM expressing low levels of BCMA obtained durable responses of greater than 2 years duration, which suggests that FHVH33-T can recognize low levels of cell-surface BCMA. Eight patients had extramedullary plasmacytomas at relapse; 4 patients had plasmacytomas biopsied. Two of the biopsied plasmacytomas were BCMA+, and two were BCMA-negative by immunohistochemistry.FHVH33-CD8BBZ CAR T cells caused relatively mild toxicity and a high rate of sCRs in patients with relapsed MM including MM with low cell-surface BCMA expression. [Display omitted] DisclosuresBrudno: Kyverna Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lam: Kite, a Gilead Company: Patents & Royalties. Kochenderfer: Kite, a Gilead Company: Patents & Royalties: on anti-CD19 CARs, Research Funding; Bristol Myers Squibb: Research Funding.

Immunophenotypic and Single-Cell Transcriptional Profiling of CD34+ Hematopoietic Stem and Progenitor Cells Mobilized with Motixafortide (BL-8040) and G-CSF Versus Plerixafor and G-CSF Versus Placebo and G-CSF: A Correlative Study of the Genesis Trial

▪Background: CD34 expression remains the most common immunophenotypic cell surface marker defining human hematopoietic stem and progenitor cells (HSPCs). Recently, use of multicolor fluorescence-activated cell sorting (mFACS) and single-cell RNA sequencing (scRNA seq) has illustrated the heterogenous nature of CD34+ HSPCs, with immunophenotypically and transcriptionally distinct subsets ranging from primitive hematopoietic stem cells (HSCs) capable of long-term multilineage potential to differentiated, lineage-committed progenitors. Meanwhile, the addition of CXCR4 inhibitors (CXCR4i) to G-CSF (G) has increased mobilization of CD34+ HSPCs for stem cell transplantation (SCT). Yet, the effect of CXCR4i +/- G on mobilization of specific immunophenotypic and transcriptional CD34+ HSPC subsets is not well-characterized. Motixafortide (M) is a novel cyclic peptide CXCR4i with a low receptor off rate and extended in vivo action vs plerixafor (Px). M was recently evaluated in the phase 3, double blind, placebo controlled GENESIS Trial as an HSPC mobilizer prior to autologous SCT (ASCT) in multiple myeloma (MM).Methods: GENESIS Trial patients were prospectively randomized (2:1) to receive either M+G or placebo (P)+G for HSPC mobilization. Demographically similar patients undergoing mobilization with Px+G prior to ASCT for MM were prospectively enrolled on a parallel tissue banking protocol. All patients received G (10 mcg/kg) on days 1-5 (and 6-8 if needed). Patients also received either M (1.25 mg/kg) or P on day 4 (and 6 if needed); or Px (0.24 mg/kg) on day 4 (and 5-7 if needed). Apheresis began day 5 (and 6-8 if needed). HSPCs were purified from apheresis product on day 5 via CD34+ immunomagnetic selection. CD34+ HSPC subset profiling was performed via mFACS and scRNA seq. CXCR4 expression and receptor occupancy was evaluated by antibody binding capacity of 12G5 and 1D9 clones.Results: Demographics were similar between the M+G (n=24), P+G (n=13) and Px+G (n=14) cohorts. By mFACS, M+G mobilized a 5.5 fold higher absolute number (#) of HSCs, multipotent progenitors (MPP) and common myeloid progenitors (CMP) vs P+G (p<0.0001); and a 2.6 fold higher # of these subsets vs Px+G (p=0.0327) (Figure 1A). M+G mobilized a 10.5 fold higher absolute # of primitive HSCs vs P+G (p<0.0001) and a 1.1 fold higher # vs Px+G (p=0.7876) (Figure 1B). M+G and Px+G mobilized a 7-8 fold higher # of plasmacytoid dendritic cell precursors vs P+G (p-values >0.05). 1D9 binding to CXCR4 on CD34+ HSPCs was similar between all 3 arms (p-values 0.45-0.75). 12G5 binding (which competes with CXCR4i's for binding to CXCR4) was significantly lower with M+G (MFI:11) vs P+G (MFI:74; p<0.0001) and Px+G (MFI:271; p<0.0001).By scRNA seq, UMAP clustering identified 3 transcriptionally similar HSC sub-clusters (HSC-I, -II and -III) mobilized by all 3 regimens; and 1 distinct HSC-PL cluster mobilized by P+G expressing heat shock protein genes (HSPA1 A/B) (Figure 1C). Differentially expressed genes (DEGs) of HSCs I-III included CD52, FTH1, HLA-E, KLF2 and LMNA. AVP was unique to HSC-I while EGR1, JUN and ZFP36 were unique to HSC-III. MPPs clustered into 3 sub-clusters (MPP-I, -II and -III). MPP-I clustered closely to HSC-II/III with low expression of genes of differentiation. MPP-II expressed DEGs (PLEK) on a continuum toward megakaryocyte-erythroid progenitors (MEP-I/II), which expressed DEGs of erythroid differentiation (HBD/HBB). MPP-I and -II contained cells from all 3 regimens. However, MPP-III was specific to M+G with DEGs (MT-ATP8, HIST1H1) associated with monocyte, lymphocyte and NK cell differentiation.Conclusions: Extended CXCR4i with M+G mobilized significantly higher #s of combined CD34+ HSCs, MPPs and CMPs vs Px+G and P+G (p-values <0.05). Additionally, M+G mobilized a 10.5 fold higher # of immunophenotypically primitive CD34+ HSCs capable of broad multilineage hematopoietic reconstitution vs P+G (p<0.0001) and similar #s vs Px+G. CXCR4 expression on CD34+ HSPCs by 1D9 binding was similar across all arms. Whereas, 12G5 binding was much lower with M+G vs P+G and Px+G, consistent with extended CXCR4 receptor occupancy by M. All regimens mobilized transcriptionally similar HSC-I-III subsets. However, lack of CXCR4i (M or Px) resulted in mobilization of more-differentiated HSCs (HSC-PL subset). Whereas extended CXCR4i with M+G (but not Px+G) mobilized a unique MPP-III subset with DEGs related to leukocyte differentiation. [Display omitted] DisclosuresCrees: BioLineRx Ltd.: Research Funding. Retting: BioLineRx Ltd.: Research Funding. Jayasinghe: WUGEN: Consultancy; MMRF: Consultancy. Vainstein: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Ickowicz: BioLineRx Ltd.: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Kadosh: BioLineRx Ltd.: Current Employment.

VarEPS: an evaluation and prewarning system of known and virtual variations of SARS-CoV-2 genomes.

The genomic variations of SARS-CoV-2 continue to emerge and spread worldwide. Some mutant strains show increased transmissibility and virulence, which may cause reduced protection provided by vaccines. Thus, it is necessary to continuously monitor and analyze the genomic variations of SARS-COV-2 genomes. We established an evaluation and prewarning system, SARS-CoV-2 variations evaluation and prewarning system (VarEPS), including known and virtual mutations of SARS-CoV-2 genomes to achieve rapid evaluation of the risks posed by mutant strains. From the perspective of genomics and structural biology, the database comprehensively analyzes the effects of known variations and virtual variations on physicochemical properties, translation efficiency, secondary structure, and binding capacity of ACE2 and neutralizing antibodies. An AI-based algorithm was used to verify the effectiveness of these genomics and structural biology characteristic quantities for risk prediction. This classifier could be further used to group viral strains by their transmissibility and affinity to neutralizing antibodies. This unique resource makes it possible to quickly evaluate the variation risks of key sites, and guide the research and development of vaccines and drugs. The database is freely accessible at www.nmdc.cn/ncovn.

Pushing the detection limits: strategies towards highly sensitive optical-based protein detection.

Proteins are one of the main constituents of living cells. Studying the quantities of proteins under physiological and pathological conditions can give valuable insights into health status, since proteins are the functional molecules of life. To be able to detect and quantify low-abundance proteins in biofluids for applications such as early disease diagnostics, sensitive analytical techniques are desired. An example of this application is using proteins as biomarkers for detecting cancer or neurological diseases, which can provide early, lifesaving diagnoses. However, conventional methods for protein detection such as ELISA, mass spectrometry, and western blotting cannot offer enough sensitivity for certain applications. Recent advances in optical-based micro- and nano-biosensors have demonstrated promising results to detect proteins at low quantities down to the single-molecule level, shining lights on their capacities for ultrasensitive disease diagnosis and rare protein detection. However, to date, there is a lack of review articles synthesizing and comparing various optical micro- and nano-sensing methods of enhancing the limits of detections of the antibody-based protein assays. The purpose of this article is to critically review different strategies of improving assay sensitivity using miniaturized biosensors, such as assay miniaturization, improving antibody binding capacity, sample purification, and signal amplification. The pros and cons of different methods are compared, and the future perspectives of this research field are discussed.

High CD52 mRNA Expression Is An Adverse Prognostic Factor In Fludarabine-Refractory CLL Treated With Alemtuzumab On The Gcllsg CLL2H Trial

Alemtuzumab is a humanized mAb that targets CD52 and has been widely used for the treatment of chronic lymphocytic leukemia (CLL) patients with refractory disease or loss / mutation of TP53. The cell-surface marker CD52 is variably expressed on peripheral blood mononuclear cells and shows relatively high expression levels on the surface of both healthy B lymphocytes and on CLL cells, but the biologic function of CD52 has not been sufficiently clarified yet.To elucidate the prognostic value of CD52 expression, we analyzed a large gene expression dataset. Gene expression profiling was performed on Affymetrix Exon 1.0 ST Arrays. Data sets were generated for 75 fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3x30 mg Alemtuzumab s.c. per week, max. 12 weeks). Normalization of the arrays was conducted by using the RMA algorithm. Statistical analysis was performed with the R software version 2.15.1.Patients were grouped into quartiles (Q1-Q4) according to CD52 expression levels (CD52 low: Q1, n=19; CD52 intermediate: Q2-Q3, n=37; CD52 high: Q4, n=19).Progression free survival (PFS) and overall survival (OS) were markedly different for patients from Q1 and Q4 (median PFS: Q1=413 days vs. Q4=145 days (p=0.0102); median OS: Q1= 879 days vs. Q4=413 days (p=0.0164)). Patients with progressive disease were overrepresented in Q4 (PD 7/18 (38.8%); ORR 5/18 (27.7%)) while rare in Q1 (PD 2/19 (10.5%); ORR 9/19 (47.3%)). Patients with low expression of CD52 had less frequently genetic/biologic and clinical characteristics of aggressive disease (Q1 vs. Q4; TP53 mutation/17p deletion: 47.36% vs. 36.84%; IGHV unmutated: 61.11% vs. 73.68%; leukocyte counts (median): 45.8 G/L vs. 55.4 G/L; thymidine kinase: 22.2 U/L vs. 30.4 U/L; β2-microglobulin: 3.3 mg/l vs. 5.2 mg/l; no palpable lymph nodes: 47.36 % vs. 10.52 %).Mutations of SF3B1 and NOTCH1 were present in 4/17 (23.5%) and in 5/17 (29.4%) cases in Q1. In contrast, cases from Q4 harbored mutations of SF3B1 and NOTCH1 in 2/18 (11.1%) and 1/18 (5.5%).Cases with high CD52 levels showed overexpression (FDR<1e-07;FC>2) of genes predominantly involved in signaling of the B-cell-, toll-like- and interleukin-receptor (BTK, SYK, CD79B; TLR7, TLR10, IL27RA, IL2RG, TRAF3IP3, TRAF5).The corresponding gene set overrepresentation analysis on KEGG pathways was significantly enriched for gene sets related to cell-extracellular matrix interaction, focal adhesion, regulation of actin cytoskeleton and cell adhesion molecules as well as associated signaling pathways like WNT- and MAPK-signaling.In conclusion, high CD52 expression is an adverse prognostic factor in refractory CLL treated with Alemtuzumab. Although suggestive, the level of CD52 expression and subsequent antibody binding capacity appear not to determine the effectivity of treatment. The association with genetic and biological characteristics indicates an independent role in the biology of aggressive CLL and highlights a potential function in cell-microenvironmental interactions for CLL cells. [Display omitted] Disclosures:Stilgenbauer:Genzyme: Honoraria, Research Funding.

CD74 Is Expressed in Relapsed and Refractory Multiple Myeloma and Can be Targeted with a Novel Anti-CD74 Antibody Drug Conjugate, SΤRO-001

BACKGROUNDCD74 is a transmembrane glycoprotein involved in MHC II protein formation and transport. STRO-001 is a novel antibody drug conjugate (ADC) comprised of a human aglycosylated anti-CD74 IgG1 antibody (SP7219) genetically incorporating the non-natural amino acid para-azidomethy-L-phenyalanine (pAMF) to enable the site-specific conjugation of a non-cleavable maytansinoid linker-warhead. Highly efficient and precise site-specific conjugation enabled by Sutro's cell-free antibody synthesis technology produced a well-defined homogeneous ADC with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on the highest in vitro and in vivo stability. METHODSBiotinylated SP7219 was used for immunohistochemistry (IHC). Bone marrow (BM) biopsy samples from patients with multiple myeloma (MM) from the UCSF Dept. of Laboratory Medicine were used after appropriate permissions were obtained. CD74 expression in plasma cells was measured as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining (Figure 1A). Plasma cells were identified based on morphology and paired CD138+ staining. Two pathologists blinded to the patient's clinical history and pathology results independently reviewed IHC specimens. SP7219 conjugated to a fluorescent dye (DBCO-Alexa647) was used for detection and quantitation of CD74 expression on MM cell lines. STRO-001 was used to determine the EC50 and the proportion of MM cells killed (span killing) in an in vitro viability assay. The anti-tumor activity of STRO-001 was evaluated in the disseminated ARP-1 and MM.1S MM models. In vivo bioluminescence imaging (BLI) for animals bearing luciferase-expressing MM.1s (MM.1s-luc) cells was performed using an IVIS Spectrum. BLI images were collected 7, 14, 21, and 28 days post-tumor cell inoculation. RESULTSOverall, CD74 expression levels were not significantly different between newly diagnosed MM (n=13) and relapsed or refractory MM (n=24) (Figure 1B). Of 37 total samples analyzed, only 1 sample lacked detectable CD74 and the majority (67%) of relapsed/refractory patients expressed CD74 levels higher than 1+ by IHC (Figure 1C). In vitro cytotoxicity assays show nanomolar potency of STRO-001 in five MM cell lines: MC/CAR (EC50 0.8 nM), ARD (EC50 ~7.0 nM), MM.1S (EC50 10-11 nM), U266B1 (EC50 8.5-9.3 nM), and ARP-1 (EC50 4.3-22 nM). CD74 cell surface expression is required for STRO-001 cytotoxic activity but expression level, measured by antibody-binding capacity, does not correlate strongly with in vitro potency (R2=0.5837 for MM cell lines). STRO-001 inhibits the growth of CD138+ plasma cells in BM and formation of visceral tumors (p=0.002 for kidney; p<0.0001 for ovary) after 4 weekly doses of 3 mg/kg in the ARP-1 disseminated MM xenograft model in severe combined immune deficient (SCID) mice. STRO-001 dosed at 3 mg/kg and 10 mg/kg weekly x 3 also eradicates malignant BM plasma cells by day 32 post-inoculation (p<0.0001) and prolongs survival in the MM.1S disseminated model in NOD SCID gamma (NSG) mice. At termination of the study, 129 days post-inoculation, 100% of the STRO-001 treated animals survived and showed no evidence of disease with no CD138+ cells in their BM, while mean survival of control animals was 35 days with almost 50% of their BM containing myeloma cells. Additionally, BLI enabled noninvasive quantitation of tumor burden in MM.1S-luc-bearing NSG mice. Single doses of 1, 3, and 10 mg/kg STRO-001 (administered on day 7 post-inoculation) resulted in eradication of myeloma by day 28 based on BLI (Figure 2A and 2B) and quantification of CD138+ cells in BM (p<.0001) (Figure 2C). CONCLUSIONCD74 expression was detected in most MM samples examined in this study. Expression is heterogeneous and observed in treatment-naïve and heavily pre-treated patients. STRO-001 demonstrates potent in vitro cytotoxicity in MM cell lines and reduces tumor burden in MM xenograft models, including prolongation of survival in the MM.1S model. Based on these encouraging observations, STRO-001 is advancing to the clinic for the treatment of myeloma and other B-cell malignancies. [Display omitted] DisclosuresEmbry:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Wiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment.

Hypomethylating Agent Leads to Enhanced Immunogenicity of a Dendritic Cell/Acute Myeloid Leukemia Fusion Vaccine and Prolonged Survival in an Immunocompetent Mouse Model

Immunotherapy for acute myeloid leukemia (AML) holds great promise as a modality to overcome tolerance and elicit memory responses needed for durable remissions. Our group pioneered a novel personalized vaccine that targets a broad array of leukemic antigens (Ags) by fusing whole patient-derived AML cells to autologous dendritic cells (DCs). In a clinical trial of AML patients in chemotherapy-induced remission, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Enhancing response to vaccination relies on increasing Ag presentation and overcoming factors in the microenvironment that contribute to tolerance.While the mechanism of action of hypomethylating agents (HMAs) is multifactorial, HMAs are increasingly recognized as exhibiting immunomodulatory functions thought to contribute to therapeutic efficacy. We have previously shown that the HMA SGI-110 augments AML Ag processing and increases AML susceptibility to T cell-mediated killing. In the present study, we aimed to elucidate the effect of HMAs on enhancing Ag presentation and modulating the AML microenvironment as well as to investigate the impact of HMA on the immunogenicity of a DC/AML vaccine.We first investigated the ability of SGI-110 to reverse critical aspects of the AML immunosuppressive milieu and enhance vaccine efficacy. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1mg/kg) to assess immunologic effects without preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). SGI-110 therapy was also associated with decreased myeloid-derived suppressor cell (MDSC) burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control.Consistent with these findings, treated mice demonstrated increased AML-specific immunity via increased T cells isolated from the BM or spleen that expressed intracellular IFNϒ following ex-vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02).We then examined the potential synergy of SGI-110 + vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNϒ in response to tumor lysate as compared to vaccine or SGI-110 alone.To further elucidate the mechanism behind the immune activating effects of HMAs, we investigated the effect of HMA on the endogenous retroviral (ERV) pathway. Activation of this pathway results in increased IFN ß expression leading to increased MHC HLA Class I expression and subsequently, immune stimulation in a solid tumor model (Chiappinelli et al. Cell . 2015). MOLM-14 cells were exposed to low-dose HMA daily X 3 days and analyzed for ERV-3 expression using western blotting (WB). The results showed significant upregulation of ERV-3 expression after treatment. Due to activation of the ERV pathway, increased IFN ß expression was observed with WB analysis. Indeed, analysis of an HLA-A2 positive human AML cell line, THP1, showed significant HLA-A2 upregulation after HMA exposure. Most importantly, HLA-A2 upregulation led to a significant rise in tumor Ag presentation, detected by measuring the binding capacity of a unique T-cell receptor-like antibody using flow cytometric analysis in THP-1 cells.Given these effects, we sought to examine whether treatment with vaccine + SGI-110 would enhance survival in this model. Mice were treated with: vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a survival advantage compared to untreated mice (p<0.05). Notably, the combination arm further prolonged survival as compared to untreated mice (p<0.05).In conclusion, SGI-110 modulates critical factors contributing to the immunosuppressive milieu in AML by decreasing T cell PD-1 expression and promoting MDSC expansion, resulting in enhanced anti-AML immunity. Moreover, SGI-110 augments the immune response to an autologous DC/AML vaccine in a murine model resulting in a survival benefit. In addition, HMA enhances Ag presentation, in part due to activation of the ERV pathway. The combination of a novel personalized DC/AML fusion vaccine and HMA holds great potential and a clinical trial is being initiated. DisclosuresAvigan:Genus Oncology: Research Funding.

The Identification of the Amino Acids on Platelet Factor 4 That Bind Pathogenic Antibodies in Heparin-Induced Thrombocytopenia

Introduction: Heparin induced thrombocytopenia (HIT) is an adverse drug reaction that occurs when heparin binds to platelet factor 4 (PF4) and forms immunogenic multimolecular complexes. As a result, anti-PF4/heparin IgG antibodies bind to the PF4/heparin complexes, leading to cross-linking of FcγIIa receptors on platelets and FcγRI on monocytes resulting in their activation and an increased risk of thrombosis. Approximately 50% of cardiac surgery patients produce anti-PF4/heparin antibodies, but only a subset (<1%) produces pathogenic HIT antibodies. Current enzyme immunoassays (EIAs) cannot distinguish between pathogenic and non-pathogenic anti-PF4/heparin antibodies and will give a positive test even in the presence of the clinically insignificant non-pathogenic HIT antibodies. Further functional testing, such as the 14C-serotonin release assay (SRA), is required to identify samples containing the pathogenic anti-PF4/heparin antibodies that will lead to HIT. In this study, we hypothesized that different epitopes on PF4 could explain some of the differences between pathogenic and non-pathogenic antibodies.Methods: We used alanine scanning mutagenesis (ASM) to produce 70 mutations of PF4, each with a unique amino acid substitution. Single point mutations were made by mutating non-alanine residues to alanine, and alanine residues to valine. The PF4 mutants were isolated from Escherichia coli and used in an EIA. The binding capacity of the monoclonal pathogenic HIT antibody (KKO) and a non-pathogenic anti-PF4/heparin antibody (RTO) to each of the PF4 mutants, relative to wild-type PF4, was determined. Point mutations of PF4 that resulted in >25% loss of binding were identified. We compared our candidate amino acids to binding sites previously identified in the PF4 and KKO crystal structure. In addition, we screened nine EIA-positive/SRA-positive (pathogenic) and six EIA-positive/SRA-negative (non-pathogenic) patient samples using the PF4 mutants. Student's t-test was used to determine whether the binding capacities of each amino acid was significantly different between the two patient sample groups.Results: We identified 12 amino acid mutations of PF4 that caused ≥25% reduction in KKO binding. Most implicated amino acids cluster in two regions within the PF4 molecule. We identified amino acids on PF4 that were required for KKO binding which were not previously identified based on crystallographic studies. These results are consistent with the previous mutagenesis and crystallographic studies (Cai et al, Nat Commun. 2015;6:8277) of the KKO and PF4 complex but also contradict some previously stated amino acids that were thought to be part of this binding site. ASM of RTO and PF4 revealed only one amino acid that was affected. We used pathogenic and non-pathogenic EIA-positive human sera to further refine the location of the binding sites. We identified 10 amino acids that were required for the binding of human pathogenic HIT sera.Conclusions: Using sequential point mutations, we identified the amino acids that are critical for the binding of pathogenic HIT antibodies to PF4. These amino acids are either directly involved in antibody binding or influence the antigenic conformation of PF4. This library of PF4 mutant proteins can help differentiate pathogenic and non-pathogenic HIT antibodies and this information can improve diagnostic assays.This study was funded by the Canadian Institutes for Health Research. DisclosuresArnold:Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Dova: Consultancy; Novartis: Consultancy, Research Funding; UCB: Consultancy; Rigel: Consultancy.